Explanation 1.CA-125 in Disease Progression and Treatment of Lymphangioleiomyomatosis Cancer antigen 125 (CA-125) is overexpressed in ovarian cancer. However, CA-125 has both poor specificity and sensitivity for detecting ovarian cancer but may be useful as a biomarker for ovarian cancer prognosis and response to therapy. We have noticed high levels of serum CA-125 in LAM patients. We hypothesized that serum CA-125 levels might be associated with disease progression in LAM patients and that sirolimus may decrease CA-125 levels. CA-125 values were in the normal range for 181 of 241 patients. LAM patients with higher serum CA-125 levels were younger and pre-menopausal. CA-125 was associated with FEV1 and DLCO. Presence of TSC or AML had no effect on serum CA-125 level. Higher serum levels of CA-125 were associated with lymphatic involvement. We measured serum CA-125 levels in patients taking sirolimus. Treatment with sirolimus was associated with decreased serum CA-125 levels. Patients with pleural effujsions showed resolution with sirolimus. VEGF-D is a biomarker used for diagnosis of LAM and response to sirolimus. When levels of VEGF-D were followed over time with sirolimus for six of the nine patients with pleural effusions and higher levels of CA-125, serum VEGF-D levels were a more informative marker than serum CA-125 levels. Immunohistochemical analysis of LAM lung tissue showed CA-125 within the LAM lung nodule adjacent to cells reactive to HMB45, a LAM cell marker, and in the bronchial epithelial cells in normal areas of LAM lung. CA-125 expression was evident in hyperplastic type II pneumocytes. The close association of CA-125 expression with HMB45-positive cells prompted us to investigate the existence of LAM cells reactive to both markers. For fluorescence microscopy, we chose anti-alpha smooth muscle actin (SMA) antibodies to identify LAM lung nodules. While HMB45 reacts mainly with the epithelioid cells of the LAM nodule, SMA is present in both epithelioid and spindle-shaped LAM cells. Confocal analysis revealed cells in the LAM lung nodule that expressed both CA-125 and SMA. CA-125 appears concentrated in a perinuclear region and SMA is a more diffuse protein. Conclusions. We have explored the clinical utility of CA-125 as a biomarker in LAM and examined the molecule in LAM tissues. We have found high levels of serum CA-125 in almost 25% of LAM patients. High levels of CA-125 were associated with premenopausal status, worse percent predicted FEV1 and the presence of pleural effusions. However, the CA-125 association with FEV1 was not a causative one. In an alternative analysis where the FEV1 was considered as an outcome, pleural effusions were a significant predictor of FEV1, while CA-125 levels were not. Levels of serum CA-125 decrease with sirolimus, perhaps reflecting both a direct effect on the LAM nodule, , and an indirect effect on pleural effusions. Some elevated CA-125 may reflect serosal membrane involvement. 2. Long-Term Effect of Sirolimus on Serum VEGF-D Levels in Patients with Lymphangioleiomyomatosis . Abnormalities in lymphangiogenesis have been recognized as a major factor in the pathogenesis of LAM. In agreement, vascular endothelial growth factor-D (VEGF-D), a growth factor that binds to vascular endothelial growth factor receptor 3, is increased in the serum of patients with LAM, especially those with lymphatic involvement. Serum VEGF-D is of value as a diagnostic biomarker of LAM. The MILES trial showed that increased serum levels of VEGF-D are associated with severity of lung disease, reduced exercise tolerance, and greater oxygen requirements. Moreover, serum VEGF-D levels were reduced by sirolimus therapy. Further, a correlation between baseline pre-sirolimus serum VEGF-D levels and changes in FEV1 was observed during sirolimus therapy. Sixty-five percent of 23 patients who exhibited a reduction in baseline serum VEGF-D levels above 42 % after 12 months of treatment with sirolimus, experienced an improvement in FEV1.. Thus, in the short-term, sirolimus reduces serum levels of VEGF-D, size of chylous effusions, lymphangioleiomyomas, and angiomyolipomas, and stabilizes lung function. We evaluated the long-term course of 25 patients with lymphangioleiomyomatosis and measured VEGF-D levels, lung function and extent of lymphatic disease before and during sirolimus therapy. Treatment with sirolimus stabilized FEV1 and DLCO over a period of 4.51.6 years, caused resolution of lymphatic disease, and reduced angiomyolipomas size and VEGF-D levels. Lower VEGF-D levels correlated with sirolimus therapy, but no significant relationship was observed between reduction in VEGF-D levels and FEV1 and DLCO during sirolimus therapy. The magnitude of VEGF-D decline was not related to the effect on lung function. Patients with lymphatic disease had higher serum VEGF-D levels, greater reduction in VEGF-D levels, and better long-term sustained improvement in lung function during sirolimus therapy, than those without lymphatic disease. We conclude that sirolimus therapy stabilizes lung function over many years of therapy while producing a sustained reduction in VEGF-D levels in patients with elevated pre-therapy levels. An association was not demonstrated between the magnitude of VEGF-D decline and the beneficial effect of sirolimus on lung function. Persistent improvement in lung function was observed in patients with lymphatic disease. Lower VEGF-D levels were associated with sirolimus therapy, older age, and absence of lymphatic disease. We found no relationship between reductions in pre-sirolimus VEGF-D levels produced by sirolimus therapy and percent-predicted FEV1 or DLCO. During sirolimus therapy there was no significant difference between menopausal and pre-menopausal patients in rates of change in FEV1 or DLCO. Great intra-individual variability in pre-sirolimus serum VEGF-D levels was observed.although levels were more uniform during sirolimus therapy. Not all patients experienced a decline in VEGF-D levels during sirolimus therapy. A reduction in VEGF-D levels was not always associated with stabilization or improvement in lung function. In patients with lymphatic involvement, complete resolution of the lymphatic pathologies was observed during sirolimus. In patients with AML, a consistent reduction in tumor volume was observed. The effect of sirolimus therapy onVEGF-D levels was different in patients with, and those without lymphatic disease. In patients with lymphatic involvement, treatment with sirolimus induced complete resolution of the lymphangioleiomyomas and chylous effusions, and reduced serum VEGF-D. In the subjects with lymphatic disease, the rate of decline of serum VEGF-D levels increased during sirolimus therapy but in those without lymphatic disease, the rate of decline of VEGF-D levels did not increase during sirolimus therapy. We found no relationship between the magnitude of VEGF-D decline and yearly rates of change of FEV1 or DLCO. Conclusions. Thus, treatment with sirolimus is effective in producing a sustained reduction in serum VEGF-D levels in LAM patients with high pre-therapy levels, which is associated with stabilization of lung function. An association between the magnitude of VEGF-D decline and a beneficial effect on lung function during sirolimus therapy was not demonstrated. Coupling of a reduction in VEGF-D levels associated with sirolimus therapy and improvement or stabilization of lung function may be seen primarily in patients with lymphatic disease involving the lung parenchyma. Lung function may continue to decline during sirolimus therapy, but this is less likely in patients with lymphatic disease.